In vitro aging of human bone marrow derived stromal cells

Mets, Tony; Verdonk, G

doi:10.1016/0047-6374(81)90035-x

Cited by 98 publications

(99 citation statements)

References 32 publications

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“…In accordance with previous studies (15,16,19,20), two major subpopulations were defined. (a) MSC with a spindle shape had two processes extending in opposite directions from an elongated cell body.…”

Section: Morphological Classificationmentioning

confidence: 81%

“…It is obvious that these cultures are composed of different cell types with distinct morphologies. Attempts have been made to classify these subpopulations by marker expression and by shape and growth kinetics, e.g., as spindle-shaped type I or flattened type II cells (19) or as rapidly self-renewing cells, or mature MSC (15,16,20). In addition to the observed heterogeneity in vitro, variations have been observed in MSC cultures obtained from different species as well as from different rodent strains or individual human donors (21)(22)(23).…”

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confidence: 99%

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Effects of plating density and culture time on bone marrow stromal cell characteristics

Neuhuber

Swanger

Howard

et al. 2008

Experimental Hematology

181

134

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Objective-Bone marrow stromal cells (MSC) are multipotent adult stem cells that have emerged as promising candidates for cell therapy in disorders including cardiac infarction, stroke and spinal cord injury. While harvesting methods used by different laboratories are relatively standard, MSC culturing protocols vary widely. This study is aimed at evaluating the effects of initial plating density and total time in culture on proliferation, cell morphology, and differentiation potential of heterogeneous MSC cultures and more homogeneous cloned subpopulations.Methods-Rat MSC were plated at 20, 200 and 2000 cells/cm 2 and grown to 50% confluency. The numbers of population doublings and doubling times were determined within and across multiple passages. Changes in cell morphology and differentiation potential to adipogenic, chondrogenic, and osteogenic lineages were evaluated and compared among early, intermediate and late passages, as well as between heterogeneous and cloned MSC populations.Results-We found optimal cell growth at a plating density of 200 cells/cm 2 . Cultures derived from all plating densities developed increased proportions of flat cells over time. Assays for chondrogenesis, osteogenesis and adipogenesis showed that heterogeneous MSC plated at all densities sustained the potential for all three mesenchymal phenotypes through at least passage 5; the flat subpopulation lost adipogenic and chondrogenic potential.Conclusion-Our findings suggest that the initial plating density is not critical for maintaining a well-defined, multipotent MSC population. Time in culture, however, affects cell characteristics, suggesting that cell expansion should be limited, especially until the specific characteristics of different MSC subpopulations are better understood. Keywordsadult stem cells; heterogeneity; variability; differentiation potential; expansion Bone marrow stromal cells (MSC) represent a heterogeneous population derived from the nonblood forming fraction of bone marrow that regulates hematopoietic cell development. In vitro, adult mesenchymal stem cells resident in this bone marrow fraction differentiate into bone, cartilage and fat (1). Cultured MSC have also been shown to regenerate cardiac (2) and Correspondence: Birgit Neuhuber, Ph.D, Department of Neurobiology and Anatomy, Drexel University College of Medicine, 2900 Queen Lane, Philadelphia, PA 19129, Phone: (215) Fax: (215) 843-9803, E-mail: bneuhuber@drexelmed.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. In this study, we cultured rat MSC for which no effort was made to limit heterogene...

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Section: Morphological Classificationmentioning

confidence: 81%

mentioning

confidence: 99%

Effects of plating density and culture time on bone marrow stromal cell characteristics

Neuhuber

Swanger

Howard

et al. 2008

Experimental Hematology

181

134

View full text Add to dashboard Cite

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“…Therefore, they are multipotential and can differentiate without cell fusion. However, the cells within the single-cell-derived colonies are morphologically heterogeneous (57) in that they contain both small, rapidly self-renewing cells (RS cells) and larger, more slowly replicating cells (44,57,(91)(92)(93). The cultures can be expanded rapidly and over one billion-fold in Ϸ8 weeks.…”

Section: Properties Of Mscsmentioning

confidence: 99%

One strategy for cell and gene therapy: Harnessing the power of adult stem cells to repair tissues

Prockop

Gregory

Spees

2003

Proc. Natl. Acad. Sci. U.S.A.

386

283

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Most recent evidence suggests that the process of tissue repair is driven by stem-like cells that reside in multiple tissues but are replenished by precursor cells from bone marrow. Among the candidates for the reparative cells are the adult stem cells from bone marrow referred to as either mesenchymal stem cells or marrow stromal cells (MSCs). We recently found that after MSCs were replated at very low densities to generate single-cell-derived colonies, they did not exit a prolonged lag period until they synthesized and secreted considerable quantities of Dickkopf-1, an inhibitor of the canonical Wnt signaling pathway. We also found that when the cells were cocultured with heat-shocked pulmonary epithelial cells, they differentiated into epithelial cells. Most of the MSCs differentiated without evidence of cell fusion but up to one-quarter underwent cell fusion with the epithelial cells. A few also underwent nuclear fusion. The results are consistent with the interesting possibility that MSCs and similar cells repair tissue injury by three different mechanisms: creation of a milieu that enhances regeneration of endogenous cells, transdifferentiation, and perhaps cell fusion.

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“…For these reasons, the cells are currently being tested for their potential use in cell and gene therapy for a number of human diseases (22,24). Previous reports (3,11,12) demonstrated that single-cell-derived colonies of human MSCs are heterogeneous in that they contain at least two morphologically distinct kinds of cells: spindle-shaped cells and large cuboidal or flattened cells. Here we have extended our previous observations (25) to demonstrate that the colonies also contain extremely small cells that are rapidly self-renewing (RS cells).…”

mentioning

confidence: 99%

“…MSCs are of interest because they are easily isolated from a small aspirate of bone marrow and readily generate single-cell-derived colonies (1-5, 25, 27); the single-cell-derived colonies can be expanded through as many as 50 population doublings in about 10 weeks (25), and they can differentiate into osteoblasts, adipocytes, chondrocytes (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13), myocytes (9), astrocytes, oligodendrocytes, and neurons (17,23,26,27). For these reasons, the cells are currently being tested for their potential use in cell and gene therapy for a number of human diseases (22,24).…”

mentioning

confidence: 99%

Identification of a subpopulation of rapidly self-renewing and multipotential adult stem cells in colonies of human marrow stromal cells

Colter

Sekiya

Prockop

2001

Proc. Natl. Acad. Sci. U.S.A.

826

675

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Marrow stromal cells are adult stem cells from bone marrow that can differentiate into multiple nonhematopoietic cell lineages. Previous reports demonstrated that single-cell-derived colonies of marrow stromal cells contained two morphologically distinct cell types: spindle-shaped cells and large flat cells. Here we found that early colonies also contain a third kind of cell: very small round cells that rapidly self-renew. Samples enriched for the small cells had a greater potential for multipotential differentiation than samples enriched for the large cells. Also, the small cells expressed a series of surface epitopes and other proteins that potentially can be used to distinguish the small cells from the large cells. The results suggested it will be important to distinguish the major subpopulations of marrow stromal cells in defining their biology and their potential for cell and gene therapy.B one marrow contains at least two kinds of stem cells, hematopoietic stem cells and stem cells for nonhematopoietic tissues (1-26), variously referred to as mesenchymal stem cells or marrow stromal cells (MSCs). MSCs are of interest because they are easily isolated from a small aspirate of bone marrow and readily generate single-cell-derived colonies (1-5, 25, 27); the single-cell-derived colonies can be expanded through as many as 50 population doublings in about 10 weeks (25), and they can differentiate into osteoblasts, adipocytes, chondrocytes (1-13), myocytes (9), astrocytes, oligodendrocytes, and neurons (17,23,26,27). For these reasons, the cells are currently being tested for their potential use in cell and gene therapy for a number of human diseases (22,24). Previous reports (3,11,12) demonstrated that single-cell-derived colonies of human MSCs are heterogeneous in that they contain at least two morphologically distinct kinds of cells: spindle-shaped cells and large cuboidal or flattened cells. Here we have extended our previous observations (25) to demonstrate that the colonies also contain extremely small cells that are rapidly self-renewing (RS cells). The RS cells appear to be the earliest progenitors in the cultures and have the greatest potential for multilineage differentiation. They differ from more mature cells in the same cultures by a series of surface epitopes and expressed proteins. MethodsIsolation and Growth of MSCs. To isolate human MSCs, bone marrow aspirates of 10-20 ml were taken from the iliac crest of normal donors ranging in age from 19 to 49 years old under an Institutional Review Board approved protocol. Nucleated cells were isolated with a density gradient [Ficoll͞Paque (Pharmacia)] and resuspended in complete culture medium [␣ MEM, GIBCO͞BRL; 20% FBS, lot-selected for rapid growth of MSCs (Atlanta Biologicals); 100 units/ml of penicillin͞100 g/ml of streptomycin͞2 mM L-glutamine (GIBCO͞BRL)]. All of the cells were plated in 25 ml of medium in a 175-cm 2 culture dish (Falcon) and incubated at 37°C with 5% humidified CO 2 . After 24 h, nonadherent cells were discarded, and adherent cells ...

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In vitro aging of human bone marrow derived stromal cells

Cited by 98 publications

References 32 publications

Effects of plating density and culture time on bone marrow stromal cell characteristics

Effects of plating density and culture time on bone marrow stromal cell characteristics

One strategy for cell and gene therapy: Harnessing the power of adult stem cells to repair tissues

Identification of a subpopulation of rapidly self-renewing and multipotential adult stem cells in colonies of human marrow stromal cells

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